Biomedical subjects

Rajesh Agarwal

Publications and source records attributed to Rajesh Agarwal.

64 records · Page 4Linked to original sources

PubMed2002

Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth Inhibition, G2-M arrest, and apoptosis.

PURPOSE: We recently demonstrated the strong anticancer efficacy of silibinin,an active constituent of a widely consumed dietary supplement milk thistle extract, against human prostate cancer cells in culture and nude mice xenografts. We also observed that pharmacologically achievable concentrations of silibinin in animal studies were in the range of 25-100 microM, depending on the dose regimen, which did not show any apparent toxicity to the animals. In this study, we assessed whether silibinin synergizes the therapeutic potential of the chemotherapeutic drug doxorubicin against prostate cancer, the effectiveness of which is limited because of high systemic toxicity. EXPERIMENTAL DESIGN: Prostate cancer cells were treated with silibinin and doxorubicin, either alone or in combination, and cell growth was determined by manual cell counting. Cell cycle progression was assessed by saponin/propidium iodide staining and fluorescence-activated cell sorter analysis. Protein levels of cell cycle regulators were determined by Western blotting, and cdc2/p34 kinase activity was analyzed by in-beads kinase assay. Apoptosis was quantified by annexin V/propidium iodide staining and fluorescence-activated cell sorter analysis. RESULTS: Silibinin strongly synergized the growth-inhibitory effect of doxorubicin in prostate carcinoma DU145 cells (combination index, 0.235-0.587), which was associated with a strong G(2)-M arrest in cell cycle progression, showing 88% cells in G2-M phase by this combination compared with 19 and 41% of cells in silibinin and doxorubicin treatment alone, respectively. The underlying mechanism of G2-M arrest showed a strong inhibitory effect of combination on cdc25C, cdc2/p34, and cyclin B1 protein expression and cdc2/p34 kinase activity. More importantly, this combination caused 41% apoptotic cell death compared with 15% by either agent alone. Silibinin and doxorubicin alone as well as in combination were also effective in inhibiting the growth of androgen-dependent prostate carcinoma LNCaP cells. CONCLUSION: These findings suggest a need for in vivo studies with this combination in preclinical prostate cancer models. Positive outcomes might be relevant for a clinical application in prostate cancer patients.

Animals↗

PubMed2002

Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention.

Several studies have identified silibinin as an anticarcinogenic agent. Recently, we showed that silibinin inhibits cell growth via G1 arrest, leading to differentiation of androgen-dependent human prostate carcinoma LNCaP cells (X. Zi and R. Agarwal, Proc. Natl. Acad. Sci. USA, 96: 7490-7495,1999). Here, we extend this study to assess the effect of silibinin on total retinoblastoma protein (Rb) levels and its phosphorylation status, levels of E2F family members, and Rb-E2F binding in LNCaP cells. Compared with controls, silibinin resulted in an increase in total Rb levels that was largely attributable to an increase in unphosphorylated Rb (up to 4.1-fold). This effect of silibinin was mainly attributable to a large decrease (70-97%) in the amount of Rb phosphorylated at specific serine sites. In other studies, silibinin showed a moderate effect on E2F1 but up to 98 and 90% decreases in E2F2 and E2F3 protein levels, respectively. Silibinin treatments also resulted in an increase in the amount of Rb binding to E2F1 (3.8-fold), E2F2 (2.2-fold), and E2F3 (2.2-fold). Cyclin-dependent kinases (CDKs), together with their catalytic subunit cyclins, phosphorylate Rb, which makes transcription factor E2Fs free from Rb-E2F complexes, resulting in cell growth and proliferation. Conversely, CDK inhibitors inhibit this phosphorylation, maintaining E2Fs bound to Rb, which causes growth inhibition. On the basis of our data showing that silibinin induces both unphosphorylated Rb levels and Rb-E2F binding, we also assessed its effect on upstream cell cycle regulators. Silibinin-treated cells showed up to 2.4- and 3.6-fold increases in Cip1/p21 and Kip1/p27 levels, respectively, and a decrease in CDK2 (80%), CDK4 (98%), and cyclin D1 (60%). Consistent with these results, silibinin showed both G1 arrest and growth inhibition. Together, these findings identify modulation of Rb levels and its phosphorylation status as a molecular mechanism of silibinin-induced neuroendocrine differentiation of human prostate carcinoma LNCaP cells and suggest that this could be a novel approach for prostate cancer prevention by silibinin.

Cell Cycle↗

PubMedDate not supplied

Inositol hexaphosphate inhibits growth and induces G1 arrest and apoptotic death of androgen-dependent human prostate carcinoma LNCaP cells.

Prostate cancer (PCA) is the most common invasive malignancy and the second leading cause of cancer-related deaths in the US male population. One approach to control this malignancy is its preventive intervention by dietary agents. Inositol hexaphosphate (IP6), a dietary constituent, has shown promising efficacy against various cancers; however, limited studies have been performed with IP6 against PCA. Here, we investigated the growth-inhibitory effect and associated mechanisms of IP6 in androgen-dependent human prostate carcinoma LNCaP cells. IP6 treatment of cells resulted in a strong growth inhibition and an increase in G1 cell population. In mechanistic studies, IP6 resulted in an increase in cyclin-dependent kinase inhibitors (CDKIs) Cip1/p21 and Kip1/p27 levels, together with a decrease in cyclin-dependent kinase (CDK) 4 and cyclin D1 protein levels. An increase in CDKI levels by IP6 also led to a concomitant increase in their interactions with CDK2 and CDK4, together with a strong decrease in the kinase activity of both CDKs. Downstream in CDKI-CDK-cyclin cascade, consistent with its inhibitory effect on CDK kinase activity, IP6 treatment of cells increased hypophosphorylated levels of retinoblastoma (Rb) with a decrease in Rb phosphorylation at serine 780, 807, and 811 sites, and caused a moderate to strong decrease in the levels of transcription factors E2F1, E2F4, and E2F5. In other studies, IP6 caused a dose- and a time-dependent apoptotic death of LNCaP cells, and a decrease in Bcl2 levels, causing a strong increase in Bax versus Bcl2 ratio, as well as an inhibition of constitutively active AKT phosphorylation. Taken together, these molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest, and apoptotic death of human prostate carcinoma LNCaP cells. Because early clinical PCA growth is an androgen-dependent response, the results of the present study employing androgen-dependent LNCaP cells suggest that IP6 has promise and potential to be effective against PCA.

Androgens↗

PubMedDate not supplied

Epidermal growth factor receptor mediates silibinin-induced cytotoxicity in a rat glioma cell line.

Silibinin, derived from milk thistle extract, has been shown to inhibit growth factor receptor-mediated mitogenic and cell survival signaling, and to alter cell cycle regulators. Alteration in pathways regulating cell growth likely account for silibinin's inhibition of tumor growth. Since the epidermal growth factor receptor (EGFR) is a key regulator in cell signaling pathways, in the present study we directly tested the hypothesis that the EGFR plays a key role in mediating silibinin cytotoxicity to cancer cells. We generated a cell line, 9L-EGFR, which stably expressed human EGFR; the parental rat glioma cell line, 9L, does not contain endogenous EGFR message or protein. Our results show that expression of EGFR was both necessary and sufficient for conferring toxicity in response to silibinin in 9L-EGFR cells. Addition of silibinin was shown to inhibit EGFR activation by EGF in 9L-EGFR cells. These studies support the hypothesis that silibinin toxicity to cancer cells involves the EGFR signaling pathway. The findings presented here provide a rationale for understanding the growth inhibition effect of silibinin in cancer cells, and warrant further investigation into the effect of silibinin on specific pathways of cell signaling mediated by the EGF receptor.

Animals↗

PubMedDate not supplied

The cancer preventive flavonoid silibinin causes hypophosphorylation of Rb/p107 and Rb2/p130 via modulation of cell cycle regulators in human prostate carcinoma DU145 cells.

Phosphorylation status of retinoblastoma (Rb) and related proteins is important to drive cell cycle progression. In hyperphosphorylated state, they are growth stimulatory, but their hypophosphorylation is growth inhibitory. Here we assessed whether silibinin causes hypophosphorylation of Rb-related proteins as its growth inhibitory response in human prostate cancer (PCA) DU145 cells. Silibinin treatment of cells resulted in a strong increase (up to 2.3-and 5.4-fold) in the levels of hypophosphorylated Rb/p107 and Rb2/p130, respectively, but a strong decrease (91, 78 and 45%) in protein levels of transcription factors E2F3, E2F4 and E2F5, respectively. In the studies analyzing whether this effect of silibinin is via modulation of cell cycle regulators, silibinin-treated cells showed a strong increase (up to 13- and 6-fold) in Cip1/p21 and Kip1/p27 levels, respectively. Silibinin treatment also resulted in 90 and 70% decrease in CDK4 and CDK2 levels, respectively, but did not alter the protein levels of cyclin D1 and cyclin E. Consistent with its effect on G1 cell cycle regulators, silibinin treated cells exhibited a strong G1 arrest, almost complete growth inhibition, and morphological changes suggestive of differentiation. Together, these results suggest that silibinin caused hypophosphorylation of Rb-related proteins may in part be responsible for its cancer preventive and anti-carcinogenic efficacy in different cancer models including PCA.

Anticarcinogenic Agents↗

PubMedDate not supplied

Phytochemicals as cell cycle modulators--a less toxic approach in halting human cancers.

The multistep nature of cancer development provides a rationale for cancer prevention. Activation of oncogenes, inactivation of tumor suppressor genes and modulation of mitogenic signal transduction pathways are critical in cancer progression and present attractive targets for cancer prevention/intervention. In this respect, cell cycle regulation and its modulation by various natural (plant-derived) and synthetic agents are gaining widespread attention in recent years. A number of phytochemicals inhibit cell cycle progression in cancer cells, yet their clinical applications are still in infancy. The present review is focused on the modulatory effects of phytochemicals on critical cell cycle molecules, and discusses how they inhibit proliferation and/or induce apoptotic death in cancer cells.

Antineoplastic Agents, Phytogenic↗

PubMedDate not supplied

Silibinin induces growth inhibition and apoptotic cell death in human lung carcinoma cells.

BACKGROUND: The high systemic toxicity of chemotherapeutic agents limits their use to treat clinical lung cancer. These limitations could be minimized/overcome by using non-toxic phytochemicals, like, silibinin. MATERIALS AND METHODS: We used small cell lung carcinoma cells (SCLC) SHP-77 and non-small cell lung carcinoma cells (NSCLC) A-549, analyzing cell growth inhibition and death with Trypan blue exclusion, indices of the cell cycle progression with flow cytometry and apoptosis with propidium iodide and Hoechst 33342. RESULTS: Silibinin (25, 50 and 100 microM) treatment of SHP-77 and A-549 cells resulted in their growth inhibition and cell death. Cell cycle studies showed a small increase in G0-G1 population at all the time intervals in SHP-77 cells, however, in A-549 cells, a slight increase in G0-G1 but strong increase in S-phases was observed at lower treatment times, and a strong increase in G0-G1 population at 72 hours. Quantitative apoptotic studies showed that silibinin causes apoptotic cell death in both a dose- and a time-dependent manner with SHP-77 cells showing more apoptotic effect than A-549 cells. CONCLUSION: Silibinin significantly induces growth inhibition, a moderate cell cycle arrest and a strong apoptotic death in both small cell and non-small cell human lung carcinoma cells, which warrants further studies to assess the efficacy of this non-toxic agent in animal lung tumor models.

Antineoplastic Agents, Phytogenic↗

PubMedDate not supplied

Inositol hexaphosphate inhibits constitutive activation of NF- kappa B in androgen-independent human prostate carcinoma DU145 cells.

BACKGROUND: One practical and translational approach to control prostate cancer (PCA) and associated death could be its preventive intervention by dietary agents. Several studies in recent years have shown both preventive and interventive efficacy of inositol hexaphosphate (IP6) in different animal tumor models, however, its effects in PCA and associated mechanisms have not been assessed in detail. Since transcription factor NF-kappa B is constitutively active in advanced and androgen-independent human PCA DU145 cells, here we assessed whether IP6 inhibits this constitutively active NF-kappa B and associated upstream effectors and whether such an affect of IP6 has biological relevance in causing inhibition of proliferation and induction of apoptosis in DU145 cells. MATERIALS AND METHODS: We used DU145 cells and assessed NF-kappa B activation by gel shift assay. Western immunoblot and in-beads kinase assay were used for protein levels and enzyme activity. Proliferation was measured by BrdU labeling and apoptosis by annexin V/PI staining followed by FACS analysis. RESULTS: Treatment of cells with 1 and 2 mM doses of IP6 resulted in a strong inhibition of NF-kappa B activation, which was consistent with a decrease in nuclear levels of NF-kappa B sub-unit proteins p65 and p50. IP6-treated cells also showed a strong inhibition in phospho-I kappa B alpha protein levels concomitant with a significant increase in total I kappa B alpha levels. This effect of IP6 also corroborated with a significant inhibition in IKK alpha kinase activity. In other studies, IP6-treated cells showed strong inhibition in cell proliferation and induction of apoptosis. CONCLUSION: Our results suggest that IP6 could be a potent dietary agent in controlling the growth of advanced PCA cells and inducing their apoptotic death, in part, by its inhibitory effect on constitutively active NF-kappa B signaling pathway.

Apoptosis↗

PubMedDate not supplied

Prostate cancer and inositol hexaphosphate: efficacy and mechanisms.

There are now extensive scientific data suggesting the potential role of dietary and non-dietary phytochemicals in the prevention and control of prostate cancer (PCA) growth and progression. PCA is a disease of elderly male populations with a relatively slower rate of growth and progression as compared to most other cancers and, therefore, is a candidate disease for preventive intervention. Overall, PCA growth and progression involve aberrant mitogenic and survival signaling and deregulated cell cycle progression, accompanied by gradual accumulation of genetic and epigenetic changes over a period of years. Several mechanisms, including overexpression of growth, survival and angiogenic factors and their receptors, together with a loss/decrease of tumor suppressor p53, retinoblastoma and cyclin-dependent kinase inhibitor, have been implicated in PCA growth and progression. Therefore, phytochemicals targeting these molecular events could have a promising role in PCA prevention and/or therapy. Inositol hexaphosphate (IP6) is a major constituent of most cereals, legumes, nuts, oil seeds and soybean. Taken orally as an over-the-counter dietary/nutrient supplement, and is recognised as offering several health benefits without any known toxicity. In vitro anticancer efficacy of IP6 has been observed in many human, mouse and rat prostate cancer cells. Completed studies also show that oral feeding of IP6 inhibits human PCA xenograft growth in nude mice without toxicity. In a recently completed pilot study, we observed similar preventive effects of IP6 on prostate tumorigenesis in the TRAMP model. Mechanistic studies indicate that IP6 targets mitogenic and survival signaling, as well as cell cycle progression, in PCA cells. IP6 is also shown to target molecular events associated with angiogenesis. Moreover, IP6 has pleiotropic molecular targets for its overall efficacy against PCA and, therefore, could be a suitable candidate agent for preventive intervention of this malignancy in humans.

Animals↗

PubMedDate not supplied

Anticancer potential of silymarin: from bench to bed side.

Silymarin consists of a family of flavonoids (silybin, isosilybin, silychristin, silydianin and taxifoline) commonly found in the dried fruit of the milk thistle plant Silybum marianum. Although silymarin's role as an antioxidant and hepatoprotective agent is well known, its role as an anticancer agent has begun to emerge. Extensive research within the last decade has shown that silymarin can suppress the proliferation of a variety of tumor cells (e.g., prostate, breast, ovary, colon, lung, bladder); this is accomplished through cell cycle arrest at the G1/S-phase, induction of cyclin-dependent kinase inhibitors (such as p15, p21 and p27), down-regulation of anti-apoptotic gene products (e.g., Bcl-2 and Bcl-xL), inhibition of cell-survival kinases (AKT, PKC and MAPK) and inhibition of inflammatory transcription factors (e.g., NF-kappaB). Silymarin can also down-regulate gene products involved in the proliferation of tumor cells (cyclin D1, EGFR, COX-2, TGF-beta, IGF-IR), invasion (MMP-9), angiogenesis (VEGF) and metastasis (adhesion molecules). The antiinflammatory effects of silymarin are mediated through suppression of NF-kappaB-regulated gene products, including COX-2, LOX, inducible iNOS, TNF and IL-1. Numerous studies have indicated that silymarin is a chemopreventive agent in vivo against a variety of carcinogens/tumor promoters, including UV light, 7,12-dimethylbenz(a)anthracene (DMBA), phorbol 12-myristate 13-acetate (PMA) and others. Silymarin has also been shown to sensitize tumors to chemotherapeutic agents through down-regulation of the MDR protein and other mechanisms. It binds to both estrogen and androgen receptors, and down-regulates PSA. In addition to its chemopreventive effects, silymarin exhibits antitumor activity against human tumors (e.g., prostate and ovary) in rodents. Various clinical trials have indicated that silymarin is bioavailable and pharmacologically safe. Studies are now in progress to demonstrate the clinical efficacy of silymarin against various cancers.

Animals↗